Attenuated measles vaccine virus strain containing specific nucleotide sequence and a method for its absolute identification

Abstract

 A measles vaccine virus AIK-C strain containing specific nucleotide sequence comprising the viral genomic RNA in the seed virus for measles vaccine or measles virus vaccine consisting of 15,894 nucleotides is provided.

Description

DETAILED EXPLANATION OF THE INVENTION

[Filed of the invention]

[0001] This invention relates to a measles virus vaccine strain comprising specific nucleotide suquence and its absolute identification method.

[Prior arts]

[0002] Measles virus is a causative virus of measles which belongs Paramyxoviridae of RNA virus.

[0003] First isolation of measles virus from patient using primary culture of human kidney cells was made by J. F. Enders et al. in 1954 ( Enders, J. F. et al. : Proc. Soc. Exp.. Biol. Med., 86 : 227 - 286, 1954 ). Attenuated measles vaccine was developed using the isolated Edmonston strain by Enders et al. ( Enders, J. F. et al : New Engl. J. Med., 263 : 153 - 159, 1960 ). The vaccine developed by Enders et al. induced, however frequently, adverse effect with pyrogenicity and exanthema. Many strains of attenuated measles virus have been established by means of further attenuation of the Edmonston strain. Among these strains the Schwarz strain established by A. J. F. Schwarz has been commonly used for live measles vaccines.

[0004] The inventors of the present invention have isolated four strains of a cold variant derived from attenuated measles virus of the Edmonston strain supplied by Dr. Enders ( Makino, S. et al., Japan. J. Microbiol., 14 : 501 - 504, 1970 ).

[0005] Reduction of immunogenicity, i. e. effectiveness according to a development of attenuation of measles virus has generally been known. Among the isolated cold variant, a viral strain which adaptively grows at 33°C, is a further attenuated measles virus with high immunogenicity having different properties as generally observed in the conventional measles virus ( Makino, S. et al., Japan. J. Microbiol., 17 ; 75 - 79, 1973 ).

[0006] In order to develop the seeds for live measles vaccines from the cold variant, an inventor of the present invention has isolated clone virus which is a strain adapted with chick embryo cells obtained from specific pathogen free eggs, having same temperature marker, and designated as AIK-C strain ( Sasaki K., Kitasato Arch. Exp. Med., 47 ; 13 - 21, 1974).

[0007] Pyrogenicity ratio ( ≦ 37.5 °C ) of AIK-C strain live vaccine produced from the seeds of AIK-C strain in measles sensitive infants is aaproximately 1/3 - 1/4 as compared with that of Schwarz strain vaccine. The AIK-C strain in a further attenuated measles virus than the Schwarz strain with the unique characteristic: having a high immunogenicity response without lowering immunogenecity ( Makino, S. et al., Kitasato Arch. Exp. Med.,47 ; 13 - 21, 1974).

[0008] Encephalitis which seems being caused by administered live measles vaccine has been observed 1 - 3 persons per one million of administered infants. None of information on such that neurological complication has been reported in spite of the administration of AIK-C strain live measles vaccine for ten million peoples in Japan. ( Hirayama, M. et al., Inf. Dis., 5 ; 495 - 503, 1983, Makino, S., ibid., 5 ; 504 - 505, 1983 ).

BACKGROUND OF THE INVENTION

[0009] Biological properties of the AIK-C strain are unique and different from the other strains of measles virus. However, the virus is easily mutated and even an attenuated measles virus strain as like the AIK-C strain, a little formation of variant strains can be observed at the growth phase. Accordingly, at the production of measles live vaccine, a quality control for identification check between the seed virus and produced vaccine virus therefrom is the most important.

MEANS FOR SOLVING THE PROBLEM

[0010] A temperature marker test for AIK-C strain has been applied as a quality control test ( Sasaki, K., Kitasato Arch. Exp. Med., 47 ; 1 - 12, 1974 ). However the said biological assay method is not always to be an absolute identification method for AIK-C strain.

[0011] Fundamental biological properties of virus depends on its gene consisting of nucleic acid of viral particles. Nucleic acid of measles virus is consisted of single strand negative RNA, and each viral strain has its own nucleic acid consisting of specific nucleotide sequence.

[0012] Complete differential identification method between viral strains is, therefore, to determine nucleotide sequence of viral nucleic acid in the strain. The inventors of the present invention has focused to the point in this respect and analysed the nucleotide sequence of nucleic acid relating to measles virus infection, thereby provided the administrative control method for stable AIK-C strain of measles virus without variant and mutation.

[0013] Hitherto known identification methods for various measles strain are a specific biological response test for virus strain, however those are not the absolute identification method. For quality control on the AIK-C strain vaccine, the aforementioned temperature marker strain vaccine, test has been applied. The inventors of the present invention has been determined whole nucleotide sequence of the AIK-C strain virus genome, in order to establish an absolute identification method of the said identification test. Since the whole nucleotide sequence of specific nucleotide sequence consisting of 15,894 bases in AIK-C strain has been clearly determined, virus can be identified at the genetic level, hence the said identification method can be the absolute determination method, so that quality control on stable AIK-C strain vaccine can easily be exactly determined. The specific nucleotide sequence of 15,894 basesis set out below as Sequence I.D. No. 1.

OBJECTS OF THE INVENTION

[0014] Therefore an object of the present invention is to provide an attenuated measles live vaccine which comprises consisting of specific nucleotide sequence.

[0015] Another object of the present invention is to provide an absolute identification method for measles virus strain.

[0016] According to the present invention, the said object can be completed by providing materials and methods as set out in the claims.

Sequence list of the above nucleotide sequence.

[0017] 

Sequence type

: nucleic acid

Strandedness

: single

Topology

: linear

Molecule type

: antigenomic DNA

Original source

Organism

: attenuated measles vaccine

Strain

: AIK-C

   According to the present invention, the said further object can be completed by providing a measles vaccine virus genomic RNA consisting of nucleotide sequence in claim 1, with partial insertion mutational sequence or defective sequence.

[0018] According to the present invention, the said still further object can be completed by providing an absolute identification method for measles vaccine virus strain which comprises detecting a specific nucleotide sequence consisting of the 15,894 nucleotides according to claim 1 in the genomic RNA of measles vaccine virus strain.

[0019] According to the present invention, the said more still further object can be completed by providing an absolute identification method according to claim 3 wherein the said identification method is detecting a part of nucleotide sequence of viral genomic RNA by Northern blot technique and polymerase chain reaction method.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Fig. 1 is a Outline of cDNA construction.

[0021] Fig. 2 is a Mapping of cDNA clones of the AIK-C.

[0022] Fig. 3 is a genomic RNA sequence of the Synthetic oligonucleotides as complementary genome RNA.

[0023] In the present invention, at first, seed virus of the measles virus AIK-C strain is used. This vaccine virus is propagated in chick embryo cells derived from specific pathogen free hen's eggs to prepare so called vaccine bulk, from which virus suspension prior art to purification can be prepared. The vaccine bulk can be a part of the virus pool prepared for the market measles AIK-C strain vaccine.

[0024] The clarified virus suspension is purified by ultra-centrifugation through continuous sucrose gradients. Virus RNA is extracted by SDS-phenol method. Synthetic primer can be synthesized with approx. 25 mer synthetic primer by amidide method.

[0025] The deoxyoligonucleotide, a synthetic primer, can be synthesized, for example, on a CYCLONE DNA SYNTHESIZER (Bioserach, Inc.).

[0026] The AIK-C virus genome RNA is used as a tempelate for the synthesis of cDNA using the above synthetic oligonucleotide primer. The AIK-C virus genome RNA is reversely transcribed by reverse transcriptase to prepare single-strand cDNA which is treated by RNase H method to prepare double-strand cDNA. The double-stranded cDNAs are treated with appropriate restriction enzymes and are inserted into the pUC plasmid ( pUC 18 or pUC 19). The above process is illustrated in Fig. 1. E. coli K 12 strain HB101 is transformed with the resulting recombinant plasmid. The transformants are selected by ampicillin resistance. Colonies containing recombinant plasmids are screened by measuring the size of palsmid DNA with agarose gel electrophoresis. To determine the AIK-C genome sequence, the cDNA are subcloned into the bacteriophage M 13 series, and the single-strand M 13 phage DNAs are isolated from candidate subclones.

[0027] Determination of the obtained cDNA clone can be made by several restriction enzyme cleavage types. In the cDNA obtained from synthetic primer such as MP-1, restriction enzyme sites of BamHI, E coRV and XbaI are located. In the plasmid extracted from ampicillin resistant strain, in case that the said type of cDNA is observed, approximately 1504 base fragments are identified by splitting with BamHI and XbaI on an agarose gel electrophoresis. Further trereatment of these fragments with EcoRV provides two fragments of 650 and 854 base fragments. Accordingly, an identification of the obtained cDNA clone has been made by cleavaging the inserted fragment in the plasmid and its size determination, or by determining the specific restriction site of measles DNA in the fragments.

[0028] To determine the base sequence, the cDNA, which are splitted by restriction enzyme to prepare cohesive end or blunt end, are inserted into M 13 phage vector in which its cloning parts are cleft by the preferable restriction enzymes. The phage holding the thus prepared recombinant DNA is infected into E. coli. Single-strand recombinant DNA is extracted from the infected phage plaques, from which the nucleotide sequence is determined by the dideoxy chain termination method ( dideoxy sequence method ).

[0029] A primer such as pMN 2, the cDNA obtained from MP-1, is splitted by BamHI and XbaI, and is cloned with pUC plasmid. After determining cDNA, DNA fragment originated from AIK-C strain by splitting with the same restriction enzyme, further the fragment is splitted by EcoRV to prepare two fragments consisting of 650 bp and 854 bp.

[0030] These fragments are legated into M 13 replicative form double-strand DNA, which is previously splitted by BamHI and XbaI, transfected into E. coli. After recombinant phage is selected, the phage is infected in E. coli and multiplied. Single-strand recombinant DNA is extracted from supernatant phase of phage solution and nucleotides sequence is determined by the dideoxy sequence method.

[0031] Virus genome of AIK-C strain has been found to be constructed by 15,894 nucleotides and its whole sequence is shown as in the sequence. A measles virus containing the said RNA genome has superior properties of AIK-C virus strain. The specific nucleotide sequence of AIK-C virus strain genome is determined by PCR method and can be compared with the virus strain whether the said strain can be used for AIK-C strain virus vaccine or whether the prepared AIK-C strain virus vaccine is strictly holding the properties of AIK-C strain. Further if measles vaccine administered infant shows exanthem and antibody of measles virus is observed in lymphocyte of the infant, the causatives can be determined by comparing with the nucleotide sequence whether the exanthem is caused by administered vaccine or not.

[0032] In the specification, following abbreviations are used.

Met ;

methionine

Ala ;

alanine

Thr ;

threonine

Leu ;

leucine

Arg ;

arginine

Ser ;

serine

Phe ;

phenylalanine

Lys ;

lysine

Asn :

asparagine

Asp ;

aspartic acid

Pro ;

proline

Ile ;

isoleucine

Gly ;

glycine

His ;

histidine

Val ;

valine

Glu ;

glutamic acid

Gln ;

glutamine

   Following examples illustrate the present invention but are not construed as limiting.

Example 1

Determination of Nucleotide sequence of a virus obtained from AIK-C virus strain vaccine bulk:

Step : a

[0033] A seed virus of measles AIK-C strain ( Seed Lot 0 - 2 ) was inoculated up to an infectivity titer of 0.05 in chick embryo cells derived from specific pathogenic free hen's egg cultured in a surface area of cell clture ( 230 cm² ) in large Roux bottle ( approx. 1 liter volume). A culture medium ( 150 ml ) of Eagle's minimum essential medium ( MEM ) containing 1 % calf serum was added therein and the virus was cultured at 33 °C.

Step : b

[0034] On third day after inoculation, cultured medium was removed, then new medium ( 150 ml ), the same as above was further added and the cultivation was continued.

Step : c

[0035] After inoculation of virus, on the sixth day, cultured was removed off. Infected chick embryo cells layer was washed three times with Hank's solution ( 100 ml ) completely. Further cultivation was continued at 33°C after adding Eagle's MEM for cell culture ( 200 ml ) containing 0.1 % sodium glutamate.

Step : d

[0036] On the tenth day after virus seed inoculation wherein specific cytopathogenic effect for measles virus was observed on whole infected cell layer, the culture medium was collected for vaccine bulk solution ( volume : approx. 200 ml, infectivity titer : 10^7.2 TCID₅₀ * /ml ) ( * medium tissue culture infective dose ).

Step : e

[0037] Vaccine bulk solution was centrifuged at 3,000 r.p.m. for 30 minutes and the supernatant solution was further centrifuged at 25,000 r.p.m. for 90 minutes. ( Beckman. L 8 - 55 M, rotor : SW 28)

Step : f

[0038] After supernatant solution was removed off, TEN buffer solution (10 mM-Tris HCl, 1 mM-EDTA, 100 mM-NaCl, pH 7.4 ) ( 1 ml ) was added to a precipitate in each centrifugal tube, and suspended well to prepare concentrated virus. The thus obtained concentrated virus suspension was adjusted to a final volume of 10 ml withadding TEN buffer solution to prepare clarified virus suspension.

Step : g

[0039] The clarified virus suspension ( 5 ml ) was layered on the 30 ∼ 60 % continuous sucrose gradients in two centrifugal tubes ( Beckman centrifuge SW 41 rotor ), prepared by 60 % ( w/v ) sucrose solution in TEN buffer solution ( 6.8 ml ) and 30 % ( w/v ) sucrose solution in TEN buffer solution (6.8 ml ) per tube, then centrifuged for 90 minutes at 207,000 g at 4 °C. Suspension was fractionated and the fractions showing highest infectivity titer ( 10^8.3 TCID₅₀/ml ) were collected and again centrifuged by the same way to prepare purified virus particles.

Step : h

[0040] The purified virus particles was diluted fivefold with TEN buffer solution, separately added each 200µl into 1.5 ml microtest tube, further added 20 % SDS ( 5 µl, phenol ( 100 µl ) and chloroform ( 100µl ) therein, then stirred completely using vortex mixer. The mixture was further centrifuged at 12,000 r.p.m. for 5 minutes to separate organic layer and aqueous layer. The aqeous layer was collected into another 1.5 ml microtest tube and treated twice with phenol extraction ( SDS-phenol extraction ). The recovered aqueous layer was mixed with 5M-NaCl (1/25 volume ), and ethanol (2.5 volume ), allowed to stand -20°C for 2 hours, then centrifuged at 12,000 r.p.m. for 10 minutes to collect precipitated RNA which was washed with 70 % ethanol and dried. The dried material was dissolved in redistilled water( 50 µl )which was sterilized autoclaving to prepare RNA suspension.

Step : i

Cyclone DNA synthesizer

[0041] Synthetic primer deoxyoligonucleotide, 12 primers comprising approx. 25 mer of MP-1 ∼ MP-11 and BEP( dT )₇ shown in Fig. 3 were synthesized by using Cyclone DNA synthesizer ( Biosearch INC. U.S.A. ).

Step : j

[0042] The RNA suspension ( 10µl ) obtained in the above Step : h ( AIK-C virus genome RNA ) was used as template for the synthesis of DNA using synthetic oligonucleotide primers ( the above synthetic DNA )( 2 µl ) of synthetic primer, MP-1 or ( MP-11 ) and cDNA was prepared by reverse transcriptase treatment. The cDNA was transferred to double-strand cDNA by using RNase H-DNA polymerase 1 ( Gubler and Hoffman, GENE 25, p 263 - 269, 1983 ).

Step : k

[0043] The obtained cDNA was cleaved in its each restriction enzyme cleavage site by BamHI-XbaI, XbaI-BamHI, BamHI-EcoRI, EcoRI-BamHI, BamHI-EcoRI, EcoRI-BglII, BglII-SacI and SacI-NcoI-XbaI. Each was inserted into a corresponding cloning site in pUC plasmid ( pUC 18 and pUC 19 ).

Step : l

[0044] E. coli HB101 was transformed with the above recombinant plasmid to obtain ampicillin resistant colonies. A plasmid DNA was extracted from the thus obtained colonies and the colonies containing recombinant plasmids were screened by measuring the size length of the fragments plasmid DNA with 0.8 % agarose gel electrophoresis.

Step : m

[0045] To obtain the 3' terminal clone of the AIK-C genome, poly ( A ) was tailed at the 3' end of the genomic RNA, an RNA suspension obtained in the above step : h, with poly ( A ) polymerase and adenosine triphosphate ( ATP ). The thus obtained 3' A tailed RNA suspension, i. e. the polyadenylated RNA was reversely transcribed using BEP ( dT )₇ primer to prepare cDNA according to the step : j. The BEP ( dt )₇ primer has the sequence ; 5'-CTGTGAATTCTGCAGGATCCTTTTTTT-3'.

Step : n

[0046] The 5' terminal clone was synthesized with the primer located close to the 5' terminus. Namely the primer containing complementary DNA in a domain of 15,592-15,615 in the measles virus genome. The synthetic primer has the sequence ; 5'-TGGAAGCTTATCCAGAATCTCAAGTCCGGCT-3'.

[0047] DNA-RNA hybrid was prepared by using the said synthetic DNA as a primer with reverse transcriptase. After alkaline treatment of the hybrid, poly ( dA ), i. e. dATP was tailed to the 3' end of the resulting cDNA with terminal deoxynucleotidyl transferase. It was subsequently converted to the double-stranded cDNA using the BEP ( dT )₇ primer in the step : m and the Klenow fragment.

Step : o

[0048] The thus obtained cDNAs were subcloned into the bacteriophage M 13 series vector ( mp 18 and mp 19 ), and the single-strand M 13 Phage DNAs were isolated. Nucleotide sequence of those cDNAs was determined with the said single-stranded DNA by means of the dideoxy chain termination method using 7-DEAZA-dGTP sequencing Kit ( Takara Shuzo ).

Step : p

[0049] Computer analysis of the nucleotide and peptide sequence were performed by GENETYX software.

Example 2

Determination of viral nucleotide of AIK-C strain seed virus grown in Vero cells ( African green monkey cell live )

Step : a

[0050] AIK-C strain seed virus ( Seed Lot No. 0 - 2 ) was inoculated in Vero cells previously cultured in large size Rouox bottle according to the method described in the Example 1, and incubated at 33 °C.

Step : b

[0051] On the day 5th of incubation, infected cell layers were washed with Hank's solution, then Eagle's MEM ( 200 ml ) without calf serum was added thereto and further incubated for 2 days. Incubated viral culture was collected to obtain virus bulk suspension ( approx. 200 ml, infective titer : 10^6.5 TCID₅₀/ml ).

Step : c

[0052] The vaccine bulk was centrifuged at 3,000 r.p.m. for 30 minu., thereafter the supernatant suspension was centrifuged at 25,000 r.p.m. for 90 min. [ Centrifuge: Beckman L8-55M, rotor : SW 28]

Step : d

[0053] After the supernatant solution was removed off, TEN buffer solution (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 7.4, 1 ml ) was added to the precipitate in each centrifugal tube, and suspended completely to prepare conc. virus material, which was adjusted to volume of 10 ml by adding TEN buffer solution to prepare starting material of virus suspension.

Step : e

[0054] The starting material of virus suspension ( 5 ml ) was layered on a 30 - 60 % continuous sucrose gradients in two tubes consisting of, in one tube, 60 % ( w/v ) sucrose solution ( TEN buffer solution ) (6.8 ml ) and 30 % ( w/v ) sucrose solution ( TEN buffer solution ) (6.8 ml ), then centrifuged at 207,000 g for 90 min. at 4 °C.

[0055] The centrifuged suspension in each tube was fractionated by means of a fraction collector and fractions showing high infectivity ( 10^8.3 TCID₅₀/ml ) were collected. The collected fraction was again centrifuged by the same manner to recover purified virus.

Step : f

[0056] The purified virus diluted fivefold with TEN buffer solution was separately set each 200 µl into 1.5 ml micro test tube. Thereto was added 20 % SDS ( 5 µl ), phenol ( 100µl ) and chloroform ( 100µl ), and completely stirred using vortex mixer. Organic layer and aqueous layer were separated by the centrifuge at 12,000 r.p.m. for 5 min. Aqueous layer was collected to another 1.5 ml micro test tube, and extracted twice with the above same SDS-phenol.

[0057] Recovered aqueous layer was mixed with 5 M NaCl ( 1/25. volume ) and ethanol ( 2.5 volume ), allowed to stand at -20°C for 2 hours, centrifuge at 12,000 r.p.m. for 10 min. to collect precipitated RNA, which was washed with 70 % ethanol, and dried. RNA suspension was prepared with sterilisation of the dried material and dissolved in redistilled water ( 50 µl ).

Step : g

Cyclone DNA synthesizer

[0058] Synthetic primer deoxyoligonucleotide, 12 primers, comprising approx. 25 mer of MP-1∼ MP-11 and BEP ( dT )₇ shown in Fig. 3 were synthesized by using cyclone DNA synthesizer ( Biosearch Inc. U.S.A. ).

Step : h

[0059] The RNA suspension ( 10 µl ) obtained in the above Example : 1 ( AIK-C virus genome RNA ) was used as atemplate for the synthesis of cDNA using synthetic oligonucleotide primers ( the above synthetic DNA ) ( 2µl of synthetic primer, MP-1 or MP-11 ) and cDNA was prepared by reverse transcriptase treatment. The cDNA was transferred to double-strand cDNA by using RNaseH-DNA polymerase I ( Gubler and Hoffman, GENE 25, p. 263-269, 1983).

Step : i

[0060] The obtained cDNA was cleaved in its each restriction enzyme cleavage site by BamHI-XbaI, XbaI-BamHI, BamHI-EcoRI, EcoRI-BamHI, BamHI-EcoRI, EcoRI-Bg1II, Bg1II-SacI and SacI-NcoI-XbaI. Each fragment was inserted into a corresponding cloning site in pUC plasmid ( pUC18 and pUC 19 ).

Step : j

[0061] E. coli HB101 was transformed with the above recombinant plasmid to obtain ampicillin resistant colonies. A plasmid DNA was extracted from the thus obtained colonies and the colonies containing recombinant plasmids were screened by measuring the size length of fragment plasmid DNA with 0.8 % agarose gel electrophoresis.

Step : k

[0062] To obtain the 3' terminal clone of the AIK-C genome, poly ( A ) was tailed at the 3' end of the genomic RNA, an RNA suspension obtained in the above step : f, with poly ( A ) polymerase and adenosine triphosphate ( ATP ). The thus obtained 3' tailed RNA suspension, i. e. the polyadenylated RNA was reversely transcribed using BEP ( dT )₇ primer to prepare cDNA according to the step : h. The BEP ( dT )₇ primer has the sequence ; 5'-CTGTGAATTCTGCAGGATCCTTTTTTT-3'.

Step : l

[0063] The 5' terminal clone was synthesized with the primer located close to the 5' terminals. Namely the primer containing complementary DNA in a domain of 15,592-15,615 in the meales virus genome. The synthetic primer has the sequence ; 5'-TGGAAGCTTATCCAGAATCTCAAGTCCGGCT-3'.

[0064] DNA-RNA hybrid was prepared by using the said synthetic DNA as a primer with reverse transcriptase. After alkaline treatment of the hybrid, poly ( dA ), i. e. dATP was tailed to the 3' end of the resulting cDNA with terminal deoxynucleotidyl transferase. It was subsequently converted to the double-stranded cDNA using the BEP ( dT )₇ primer in the step : k and the Klenow fragment.

Step : m

[0065] The thus obtained cDNA were subcloned into the bacteriophage M 13 series vector ( mp 18 and mp 19 ), and the single-stranded M 13 phage DNAs were isolated. Nucleotide sequence of those cDNAs was determined with the said single-stranded DNA by means of the dideoxy chain termination method using 7-DEAZA-dGTP sequencing kit ( Takara Shuzo ).

Step : n

[0066] Computer analysis of the nucleotide and peptide sequences were performed by GENETYX software.

Example 3

Identification of measles virus by PCR method obtained from a patient of mesles appeared on the day 1

Step : a

[0067] Blood specimen ( approx. 5 ml ) was collected from a patient who appeared measles exanthem on the first. Lymphocytes were separately by Ficoll ( trade name).

Step : b

[0068] The lymphocytes were washed twice with PBS. A denaturation solution D ( 4M guanidium thiocyanate, 25 mM sodium citrate, pH 7.5 ; 0.5 % sacrosine, 0.1 M 2-mercapto ethanol, 200 µl ) was added thereto. After gently stirred, 2 M sodium acetate ( 20µl, pH 4 ), phenol ( 200 µl ) and chloroform ( 100 µl ), were added in this order.

[0069] The mixutre was treated with vortex mixer for 10 sec. and allowed to set in ice-water for 15 min. Then the mixture was centrifuged at 10,000 g for 20 min. to recover aqueous layer. Equal volume of isopropanol was added therein, allowed to stand at -20 °C for one hour, then centrifuged at 10,000 g for 20 min. to precipitate RNA. [ Chomczynski and Sacchi, Anal. Biochem., 162, 156-159 ( 1987) ].

Step : c

[0070] The thus obtained RNA was treated with reverse transcriptase reaction and PCR method.

Step : d

[0071] Nucleotide sequencing and identififcation of measles virus AIK-C vaccine strain and wild strain performed.

Claims

1. A cDNA having the nucleotide base sequence set out in Sequence I.D. No. 1.

2. An isolated cDNA according to claim 1.

3. A RNA having a nucleotide base sequence corresponding to that set out in Sequence I.D. No. 1.

4. An absolute identification method for measles vaccine virus strain which comprises detecting a specific nucleotide sequence consisting of 15,894 nucleotides in a sequence corresponding to the DNA sequence set out in Sequence I.D. No. 1, in the genomic RNA of the measles vaccine virus strain.

5. A method according to claim 4 comprising detecting a part of the nucleotide sequence of the viral genomic RNA by the Northern blot technique and the polymerase chain reaction method.

6. A method according to claim 4 comprising detecting a specific DNA sequence consisting of the 15,894 nucleotides set out in Sequence I.D. No. 1.

7. A measles virus vaccine strain containing a specific nucleotide sequence comprising the whole viral genomic RNA, in the seed virus and measles virus vaccine, consisting of 15,894 nucleotide bases in a sequence corresponding to the DNA base sequence set out in Sequence I.D. No. 1.

8. A measles vaccine virus comprising a viral genomic RNA consisting of a nucleotide sequence corresponding to the DNA sequence set out in Sequence I.D. No. 1 with partial insertion mutational sequence or defective sequence.

Drawing